AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究

应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼中克隆得到编码液泡膜Na /H 逆向转运蛋白的AtNHX1基因cDNA 编码ORF.并在该ORF上游分别插入CaMV 35启动子和TMV RNA5'UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达栽体pNT质粒的T-DNA区构建了携带AtNHX1 基因的植物表达载体质粒pNT-AtNHX1.将pNT-AtNHX1 导入农杆菌LBA4404,用农杆菌介导法将AtNHX1 基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株.通过对农杆菌茵液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系.经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1 基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录.野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织.施加梯度NaCl胁迫后,植株叶片K ,Na 含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na 和K ,维持较高的K /Na ;转基因株系叶片相对电导率显著低于野生型.上述结果表明,AtNHX1 基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害.关键词: AtNHX1 草木樨状黄芪农杆菌遗传转化耐盐性.     

Involvement of hydrogen peroxide and nitric oxide in salt resistance in the calluses from Populus euphratica

Nitric oxide (NO) and hydrogen peroxide (H2O2) function as signalling molecules in plants under abiotic and biotic stresses. Calluses from Populus euphratica, which show salt tolerance, were used to study the interaction of NO and H2O2 in plant adaptation to salt resistance. The nitric oxide synthase (NOS) activity was identified in the calluses, and this activity was induced under 150 mM NaCl treatment. Under 150 mM NaCl treatment, the sodium (Na) percentage decreased, but the potassium (K) percentage and the K/Na ratio increased in P. euphratica calluses. Application of glucose/glucose oxidase (G/GO, a H2O2 donor) and sodium nitroprusside (SNP, a NO donor) revealed that both H2O2 and NO resulted in increased K/Na ratio in a concentration-dependent manner. Diphenylene iodonium (DPI, an NADPH oxidase inhibitor) counteracted H2O2 and NO effect by increasing the Na percentage, decreasing the K percentage and K/Na ratio. NG-monomethyl-L-Arg monoacetate (NMMA, an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO, a specific NO scavenger) only reversed NO effect, but did not block H2O2 effect. The increased activity of plasma membrane (PM) H+ -ATPase caused by salt stress was reversed by treatment with DPI and NMMA. Exogenous H2O2 increased the activity of PM H+ -ATPase, but the effect could not be diminished by NMMA and PTIO. The NO-induced increase of PM H+ -ATPase can be reversed by NMMA and PTIO, but not by DPI. Western blot analysis demonstrated that NO and H2O2 stimulated the expression of PM H+ -ATPase in P. euphratica calluses. These results indicate that NO and H2O2 served as intermediate molecules in inducing salt resistance in the calluses from P. euphratica under slat stress by increasing the K/Na ratio, which was dependent on the increased PM H+ -ATPase activity.     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.     

Enhanced sensitivity to oxidative stress in an Arabidopsis nitric oxide synthase mutant

The possible involvement of nitric oxide (NO) in oxidative stress tolerance was studied using Arabidopsis thaliana wild type (WT) and Atnos1 mutant plants, in which endogenous NO production is greatly diminished because 80% of nitric oxide synthase (NOS) activity is eliminated due to T-DNA insertion in the first exon of the NOS1 gene. Compared with WT, Atnos1 mutant plants showed increased hypersensitivity to salt stress and methyl viologen (MV) treatment. The maximal photochemical efficiency of photosystem II (F(v)/F(m)) and membrane integrity decreased in WT and Atnos1 mutant plants under stresses, but the extent was higher in the mutant. Treatment with sodium nitroprusside (SNP) (a NO donor) to Atnos1 mutant plants alleviated the damage. Instead, inhibition of nitric oxide accumulation in the WT plants produced opposite effects. Hydrogen peroxide and lipid peroxidation increased and the extent was higher in Atnos1 mutant plants than that in WT plants under MV stress. These results indicated that nitric oxide could protect the damage against NaCl and MV treatments.     

Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis

Regulating the intracellular Na+/K+ ratio is an essential process for salinity tolerance. The yeast mutant, can, which is deficient in calcineurin, can not grow on medium containing Na+ because it is unable to regulate the intracellular Na+/K+ ratio. Expression of the STO gene of Arabidopsis thaliana in the can mutant complements the salt-sensitive phenotype. A protein of Arabidopsis, an H-protein promoter binding factor (HPPBF-1), that binds to STO protein was isolated. HPPBF-1 cDNA has a sequence encoding a Myb DNA binding-motif and its gene expression is induced by salt stress. Furthermore, HPPBF-1 protein is localized in the nucleus. Although, the expression level of STO is not induced under salt-stress conditions, overexpression of STO in a transgenic Arabidopsis plant gave it a higher salt tolerance than was observed in the wild type. When STO transgenic plants and wild-type plants were subjected to salt stress, root growth was increased by 33-70% in the transgenic plants under salt stress. These results suggest that STO is involved in salt-stress responses in Arabidopsis.     

The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type. There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot. We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem.     

超表达AVP1基因提高转基因百脉根的耐盐性和抗旱性

本研究以超表达拟南芥液泡膜H＋-焦磷酸酶编码基因AVPI的转基因百脉根为材料,对其耐盐性和抗旱性进行了检测。结果显示：在200mmol·L^-1 NaCl下处理或自然干旱7d后,转基因植株的生长虽然受到抑制,但受抑程度明显低于野生型植株,前者叶片相对含水量比后者分别高18％和14％,净光合速率分别高20％和21％,而MDA含量则分别低35％和27％,相对质膜透性分别低28％和27％。此外,随着盐和干旱胁迫的加剧,与野生型植株相比,转基因植株体内积累了更多Na＋、K＋和Ca2＋。以上结果表明,AVPI基因的超表达可能提高了百脉根细胞Na＋区域化能力,既减轻了过量Na＋对细胞质的毒害作用,也提高了植株的渗透调节能力,从而增强了百脉根的耐盐性和抗旱性。     

Protection of plasma membrane K+ transport by the salt overly sensitive1 Na+-H+ antiporter during salinity stress

Physicochemical similarities between K(+) and Na(+) result in interactions between their homeostatic mechanisms. The physiological interactions between these two ions was investigated by examining aspects of K(+) nutrition in the Arabidopsis salt overly sensitive (sos) mutants, and salt sensitivity in the K(+) transport mutants akt1 (Arabidopsis K(+) transporter) and skor (shaker-like K(+) outward-rectifying channel). The K(+)-uptake ability (membrane permeability) of the sos mutant root cells measured electrophysiologically was normal in control conditions. Also, growth rates of these mutants in Na(+)-free media displayed wild-type K(+) dependence. However, mild salt stress (50 mm NaCl) strongly inhibited root-cell K(+) permeability and growth rate in K(+)-limiting conditions of sos1 but not wild-type plants. Increasing K(+) availability partially rescued the sos1 growth phenotype. Therefore, it appears that in the presence of Na(+), the SOS1 Na(+)-H(+) antiporter is necessary for protecting the K(+) permeability on which growth depends. The hypothesis that the elevated cytoplasmic Na(+) levels predicted to result from loss of SOS1 function impaired the K(+) permeability was tested by introducing 10 mm NaCl into the cytoplasm of a patch-clamped wild-type root cell. Complete loss of AKT1 K(+) channel activity ensued. AKT1 is apparently a target of salt stress in sos1 plants, resulting in poor growth due to impaired K(+) uptake. Complementary studies showed that akt1 seedlings were salt sensitive during early seedling development, but skor seedlings were normal. Thus, the effect of Na(+) on K(+) transport is probably more important at the uptake stage than at the xylem loading stage.     

Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia

Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.     

等渗Ca(NO3)2和NaCl胁迫对黄瓜幼苗生长及渗透调节物质含量的影响

采用营养液培养方法,以耐盐性较弱的‘津春2号’黄瓜品种为试材,研究了等渗Ca(NO3)2和NaCl胁迫对黄瓜幼苗生长、根系电解质渗透率、根系活力、Na+和K+含量及渗透调节物质含量的影响。结果显示:(1)在84mmol.L-1 NaCl和56mmol.L-1 Ca(NO3)2等渗胁迫下,黄瓜幼苗鲜重和干重均显著下降,且NaCl处理下降的幅度大于等渗Ca(NO3)2处理。(2)NaCl主要通过对黄瓜根系的伤害来抑制植株生长,表现为根系活力下降、根系质膜透性增大、Na+大量积累、K+含量显著下降、Na+/K+明显上升,最终导致根冠比下降;而Ca(NO3)2处理对根系质膜透性、K+含量、Na+/K+的影响均小于NaCl胁迫,且根系活力和根冠比上升,但Ca(NO3)2胁迫后叶片含水量和渗透调节能力均小于NaCl胁迫。(3)NaCl胁迫条件下,黄瓜幼苗内渗透调节物质以可溶性糖为主,而Ca(NO3)2胁迫以可溶性蛋白为主。研究表明,NaCl胁迫对黄瓜幼苗的伤害大于等渗Ca(NO3)2,NaCl主要通过破坏根系质膜结构影响植株生长,而Ca(NO3)2主要通过引起地上部生理干旱来影响植株生长。     

The cyclic nucleotide-gated channel, AtCNGC10, influences salt tolerance in Arabidopsis

Cyclic nucleotide-gated channels (CNGCs) in the plasma membrane transport K+ and other cations; however, their roles in the response and adaptation of plants to environmental salinity are unclear. Growth, cation contents, salt tolerance and K+ fluxes were assessed in wild-type and two AtCNGC10 antisense lines (A2 and A3) of Arabidopsis thaliana (L.) Heynh. Compared with the wild-type, mature plants of both antisense lines had altered K+ and Na+ concentrations in shoots and were more sensitive to salt stress, as assessed by biomass and Chl fluorescence. The shoots of A2 and A3 plants contained higher Na+ concentrations and significantly higher Na+/K+ ratios compared with wild-type, whereas roots contained higher K+ concentrations and lower Na+/K+ ratios. Four-day-old seedlings of both antisense lines exposed to salt stress had smaller Na+/K+ ratios and longer roots than the wild-type. Under sudden salt treatment, the Na+ efflux was higher and the K+ efflux was smaller in the antisense lines, indicating that AtCNGC10 might function as a channel providing Na+ influx and K+ efflux at the root/soil interface. We conclude that the AtCNGC10 channel is involved in Na+ and K+ transport during cation uptake in roots and in long-distance transport, such as phloem loading and/or xylem retrieval. Mature A2 and A3 plants became more salt sensitive than wild-type plants because of impaired photosynthesis induced by a higher Na+ concentration in the leaves.     

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.     

Role of nitric oxide under saline stress: implications on proline metabolism

The present work is focused on the possible relationship between nitric oxide and the induction of proline in response to salt stress. The plants were subjected to 100 mM NaCl and sodium nitroprusside (SNP; the donor of NO) at different concentrations. The plants showed lower NaCl-induced oxidative stress and proline accumulation after application of low concentrations of SNP together with the NaCl treatment. The reduction in the proline content was related to increased activity of proline dehydrogenase. These results suggest that the NO could be capable of mitigating damage associated with salt stress.     

NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na⁺/K⁺homeostasis in Arabidopsis under salt stress

Maintaining cellular Na(+)/K(+) homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na(+)/K(+) homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na(+) contents, lower K(+) contents, and resultant greater Na(+)/K(+) ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H(2)O(2) can partially reverse the increased effects of NaCl on Na(+)/K(+) ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na(+)/K(+) ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K(+) currents were arrested, and NaCl-promoted increases in cytosolic Ca(2+) and plasma membrane Ca(2+) influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na(+)/K(+) homeostasis, thus improving the salt tolerance of Arabidopsis.